Template Switching Oligonucleotide LNA Modification for Single-Cell cDNA Synthesis

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Solution Overview

Problem

Current methods for single-cell gene expression analysis face challenges in obtaining full-length cDNA coverage from single-cell RNA amounts, often sacrificing either coverage, sensitivity, or throughput, with existing methods like Smart-Seq relying on template switching being inefficient for improving cDNA library yield and average length.

Innovation Solution

The method involves using a template switching oligonucleotide with a locked nucleic acid residue and optimizing conditions with additives like betaine and increased MgCl2 concentrations, along with modifying PCR preamplification protocols to enhance cDNA synthesis and yield from single cells.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If template switching is used for single-cell cDNA synthesis, then full-length coverage is improved, but cDNA library yield and average length remain insufficient

Engineering Contradiction:

Improvefull-length coverageVSAvoidcDNA library yield

Core Design Contradiction:

Manufacturing precisionVSQuantity of substance

Solution Approach 1:

The patent modifies the template switching oligonucleotide (TSO) sequence by incorporating locked nucleic acid (LNA) residues, which changes the chemical and thermal parameters of the oligonucleotide. This enhancement improves reverse transcription efficiency and template switching reaction efficiency, thereby increasing both cDNA library yield and average length while maintaining full-length coverage

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent uses a composite approach by combining LNA-modified TSO with specific buffer conditions (betaine, MgCl2 concentrations) and optimized PCR preamplification protocols. This composite system works synergistically to improve both the quantity and quality of cDNA libraries from single cells

Inventive Principle:

Principle #40Composite materials

2Quantity of substance

If existing template switching methods are used, then some cDNA is obtained, but the average length and yield are insufficient for effective sequencing

Engineering Contradiction:

ImprovecDNA yieldVSAvoidcDNA average length

Core Design Contradiction:

Quantity of substanceVSLength of stationary object

Solution Approach 1:

By changing the TSO sequence parameters to include LNA residues and optimizing reaction conditions (betaine concentration, MgCl2 levels), the patent simultaneously improves both cDNA yield and average length, overcoming the limitation of existing methods that produced insufficient amounts of short cDNA fragments

Inventive Principle:

Principle #35Parameter changes

3Adaptability or versatility

If single-cell RNA amounts are used, then cellular heterogeneity analysis is enabled, but full-length cDNA coverage is difficult to obtain

Engineering Contradiction:

Improvesingle-cell analysis capabilityVSAvoidfull-length coverage

Core Design Contradiction:

Adaptability or versatilityVSManufacturing precision

Solution Approach 1:

The LNA-containing TSO and optimized reaction parameters enable efficient reverse transcription and template switching from extremely low input amounts (single-cell RNA), achieving both single-cell adaptability and full-length coverage that was previously difficult to obtain

Inventive Principle:

Principle #35Parameter changes

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This approach significantly increases cDNA library yield and average length, improving sensitivity and accuracy of single-cell transcriptome analysis, reducing bias, and making the process more cost-effective and amenable to automation.

Implementation Method 1

a template switching oligonucleotide (TSO) having a sequence of AAGCAGTGGTATCAACGCAGAGTACrGrG+N, wherein +N is a locked nucleic acid (LNA) nucleotide residue

Methodology Applied

Scientific EffectLocked nucleic acid (LNA) binding:

Implementation Method 2

conducting a reverse transcriptase reaction by contacting said RNA-cDNA intermediate with a template switching oligonucleotide

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Data Source

PatentEP3036336B1Methods and compositions for cdna synthesis and single-cell transcriptome profiling using template switching reaction

Publication Date: 2020.09.30 LUDWIG INSTITUTE FOR CANCER RESEARCH LTD

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Figure 2A~2F
Figure 3~4B

AI summary

This application discloses methods for cDN'A synthesis with improved reverse transcription, template switching and preamplification to increase both yield and average length of cDNA libraries generated from individual cells. The new methods include exchanging a single nucleoside residue for a locked nucleic acid (INA) at the TSO 3' end, using a methyl group donor, and/or a MgCb concentration higher than conventionally used. Single-cell transcriptome analyses incorporating these differences have full-length coverage, improved sensitivity and accuracy, have less bias and are more amendable to cost-effective automation. The invention also provides cDNA molecules comprising a locked nucleic acid at the 3'-end, compositions and cDNA libraries comprising these cDNA molecules, and methods for single-cell transcriptome profiling.